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Ellen Townes-Anderson, Jianfeng Wang, Eva Halasz, Ilene Sugino, Amy Pitler, Marco A Zarbin; Fasudil, a clinically used ROCK inhibitor, stabilizes photoreceptor synapses after retinal detachment. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5965.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal detachment causes the disjunction of the rod-bipolar synapse in the outer plexiform layer (OPL) by retraction of rod axons. In animal models, reattachment does not restore broken synapses fully. We have determined that breakage is due to activation of the RhoA pathway and that inhibition of RhoA signaling, using the Rho kinase (ROCK) inhibitor Y27632, reduces retinal disruption (Wang et al. IOVS 2016). Here we test whether the ROCK inhibitor fasudil, used for other clinical applications, can prevent synaptic injury after detachment.
Retinal explants, detached from the retinal pigmented epithelium, were maintained in culture for 2 hrs and then subjected to western blotting. In vivo, detachments were made in young Yorkshire pigs by subretinal injection of BSS in the inferior nasal retina. Fasudil (1,10mM) was injected either subretinally or intravitreally at the time of detachment. After 2 hrs, retinas were fixed for examination by immunocytochemistry and confocal microscopy. Retraction of rod terminals was quantified by imaging the amount of synaptic vesicle label in the outer nuclear layer (ONL); normally label remains exclusively in the OPL. Additionally, retinas were analyzed for apoptosis in the ONL using propidium iodide staining.
Phosphorylated cofilin, a downstream effector of ROCK, was decreased by 26% with 30µM fasudil in vitro (N=5 explants, p<0.05); total cofilin remained unchanged. In vivo, subretinal injection of fasudil (10mM) reduced retraction of rod spherules by 59% compared to untreated control detachments (N=3 pigs, p<0.001, paired t-test). This compares well with previous results with Y27632 (Wang et al.). Intravitreal injection of 10mM fasudil, potentially a more clinically relevant route of administration, also reduced retraction of rod axons by 34% (N=5, p<0.05). Controls had no evidence of photoreceptor degeneration at 2 hrs, but by 4 hrs apoptosis was evident. With 10mM fasudil the number of apoptotic nuclei was substantially reduced at 4 hrs (51% reduction, N=4, p<0.05).
Fasudil reduced photoreceptor degeneration and preserved the rod-bipolar synapse after retinal detachment. These results add additional support to the possibility, previously tested with Y27632, that ROCK inhibition may increase visual restoration after detachment when combined with surgical reattachment.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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